Methods Relating to Tissue Culture of G1E Cells

G1E cells are a GATA- (null) cell line derived from targeted disruption of GATA-1 in embryonic stem cells. These cells propagate in culture with a doubling time of about twelve hours. They resemble proerythroblasts and do not differentiate. They are dependent on Kit and Erythropoietin, but not IL-3. In our studies of erythroid differentiation, we have employed a G1E subclone designated G1E-ER4 which stably expresses a fusion product combining GATA-1 with the estradiol receptor ligand binding domain. Addition of either estradiol or taxoxifen (4-OHT) to the medium results in the functional activation of GATA-1. This triggers erythroid differentiation which becomes morphologically apparent at about 12 hours. By twenty-four hours, hemoglobin can be detected by benzidine staining. Microarray analysis indicates that transcriptional changes take place within the first hour of GATA-1 activation, however. By 30 hours, G1E cells reach a stage developmentally equivalent to an orthrochromatic erythroblast. They continue to hemoglobinize up to 72 hours, but then undergo apoptosis. They do not extrude their nuclei. Cells remain in log phase when maintained between 1 x 105 and 1 x 106 cells/mL; effectively this means passaging daily with splits between 1:4 and 1:10.

G1E Medium

Component

Store At

Volume

Iscove’s MDM

4C

500 mL

FCS (ES-grade)

-20C

75 mL

Penicillin/Streptomycin stock

-20C

10 mL

Monothioglycerol (MTG)

4C

6.2 uL

Kit-ligand conditioned medium

-20C

3 mL

Erythropoetin 10,000 U/mL

-20C

100 uL

Puromycin is maintained as 1000X stock at -20C and can be added directly to culture flasks to maintain selection pressure on GIE-ER4. When a new batch is thawed, it is routine passed for one or two generations in media containing puromycin to select against revertants lacking GATA-1-ER.

Freezing cells

2x Freezing Medium

Component

Store At

Volume

FCS

-20C

4 mL

DMSO

Room Temp, Dark

1 mL

Mix well, keep cold.

Good for a few days at 4C.

Make 2X medium and keep on ice. Fill freezing container with isopropanol and prechill to 4C.

Count cells; should be in log phase 0.5 - 0.8 x 106/mL

Calculate volume necessary for n number of 1 mL vials with 5-10 x 106 per vial.

Quickly place vials into freezing container. Put freezing container in -80C freezer overnight. The next day, transfer to liquid nitrogen.

Cell Staining

Benzidine Staining (to assess hemoglobin content)

Dissolve 60 mg of o-diansidine (Sigma D-9143) in 29.7 mL H2O and 0.5 mL glacial acetic acid. Note o-diansidine is a toxic carcinogen; wear gloves and work in fume hood. To dissolve, heat gently and stir for 30-60 minutes. Centrifuge to eliminate undissolved particulate matter. Store supernatant at 4C in light-proof container. Reagent is good for several months, but should be discarded when it turns brown.

Add one part of above preparation to 10 parts of the media containing cells; pH change turns media yellow-green.

After 1-2 minutes, observe with phase contrast microscope with filter removed, or on hemocytometer, a wet-prep slide or in the flask itself. Positive cells are red; negative are clear. Induced GIE and MEL are positive controls. About 0.05-0.1% of uninduced MEL cells are positive. After 10 minutes, large air bubbles make observation difficult.

To create slides

Dilute one part of above preparation into 10 parts media containing cells. Ideally, 2-5 x 105/mL in 50 to 100 uL. Do not exceed 150 uL.